Progressive Design of a Ranatuerin-2 Peptide from Amolops wuyiensis: Enhancement of Bioactivity and In Vivo Efficacy

Antimicrobial peptides (AMPs) that exert multiple functions are considered promising candidates to combat the bacterial drug resistance crisis. Nowadays, targeted peptide modification has been widely recognised to improve biological activity and make up for deficiencies in clinical applications such as toxicity. In this study, a helix-loop peptide was isolated and identified from the skin secretion of the Wuyi torrent frog Amolops wuyiensis, namely, ranatuerin-2-AW (R2AW) (GFMDTAKNVAKNVAATLLDKLKCKITGGC). Target modifications were made to R2AW to study the structure–activity relationships and to optimise its bioactivities. Five analogues were progressively designed via residue substitution and truncation and the antibacterial and anticancer activities were evaluated. We found that the serine-substitution and cyclic-domain-deletion products showed similar antibacterial activity to the natural peptide R2AW, implying that the disulphide bridge and Rana box were dispensable for the antibacterial activity of ranatuerin-2 peptides. Notably, the cationicity- and hydrophobicity-enhanced variant, [Lys4,19, Leu20]R2AW(1-22)-NH2, exhibited significantly optimised antibacterial and anticancer activities. Additionally, it killed bacteria by membrane disruption at a highly efficient rate. Moreover, [Lys4,19, Leu20]R2AW(1-22)-NH2 exerted potential in vivo efficacy in a methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Overall, this study demonstrated some rational design ideas for optimising the dual antibacterial and anticancer activities of ranatuerin-2 peptides and it proposes [Lys4,19, Leu20]R2AW(1-22)-NH2 as an appealing candidate for therapeutic development.


Introduction
The high mortality rates and economic burden caused by drug-resistant pathogens are considered among the most serious health threats worldwide [1,2].Recent statistics have revealed that infection mortality is on an increasing trend with limited novel therapies available [3].Another challenge is the increasing incidence of cancer.According to one report, there were approximately 18.3 million new cancer cases globally in 2018, with lung, breast, prostate, and colorectal cancer accounting for most diagnosed cases [4,5].Moreover, researchers have pointed out that chronic infections are related to cancer [6][7][8].Currently, commercially available drugs for the treatment of infection and cancer have low selectivity and have induced severe drug resistance.Therefore, it is urgent to discover new agents that can kill or inhibit the growth of different microorganisms and cancer cells.
Accordingly, the structures of more than 3000 antimicrobial peptides (AMPs) are currently archived in the antimicrobial peptide database (APD3) [9].AMPs from amphibian skin secretions are generally characterised by having cationic and amphipathic properties and may play important roles in host defence systems.As stated, AMPs possess antibacterial and antifungal activities and some also have shown significant anticancer activities [9,10].In general, the membrane-targeting mechanism of AMPs can be explained by models, such as pore and carpet models.The advantages of rapid killing mechanisms and less possibility of drug resistance induction make AMPs a most promising candidate group for evaluation to solve the crisis [11].
AMPs from Ranidae frogs have significant structural diversity and have been divided into several families, including brevinins and ranatuerins [12].Ranatuerin-2 was first isolated and identified from the skin secretion of Lithobates catesbeianus and is commonly found in North American frogs and the Chinese bamboo leaf odorous frog [13,14].The primary structures of ranatuerin-2 peptides are poorly conserved, characterised by several residue deletions and two invariant cysteines forming the cyclic hexapeptide or heptapeptide domain (Rana box) at the C-terminus [15].In general, peptides from the ranatuerin-2 family show a broad-spectrum activity against bacteria with low haemolysis [12].As well as this, peptides such as ranatuerin-2PLx can prevent cancer cell proliferation, indicating the dual therapeutic potential of these peptides [16].Hence, this small peptide with low toxicity and lack of drug resistance has excellent potential and value to be developed into a dual antibacterial and anticancer agent.
Generally, the comprehensive study of structure-activity relationships will contribute to the rational design of AMPs as a substitute for traditional agents for clinical applications.So far, the role of the distinct cyclic domain, the Rana box, in helix-loop AMPs remains ambiguous.As reported, the C-terminal heptapeptide domain is crucial for maintaining the bioactivities for B1CTcu5, whereas it is dispensable for nigrocin-HL [17,18].However, the functions of the Rana box and the disulphide bridge in ranatuerin-2 peptides have not been clarified clearly.
In this study, a ranatuerin-2 peptide (R2AW) was isolated and identified from the skin secretion of Amolops wuyiensis and the roles of the cyclic heptapeptide domain and the cysteine bridge in the peptide were studied.Apart from the study of the structure-activity relationships, this work focused on specific modifications of peptides to optimise their antibacterial and anticancer activities.Hence, a series of peptides were chemically synthesised by use of a solid-phase peptide synthesiser.After that, reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionisation timeof-flight (MALDI-TOF) mass spectrometry were applied for peptide purification and identification.The in vitro antibacterial and antibiofilm abilities were evaluated using different bacterial strains.The antiproliferative effect was assayed on human cancer cell lines.Additionally, the haemolytic effect was assessed on horse red blood cells.Moreover, the in vivo antibacterial efficacy was examined using a methicillin-resistant Staphylococcus aureus (MRSA)-infected larvae model.

"Shotgun" Cloning of R2AW from Amolops Wuyiensis Skin Secretion-Derived cDNA Library
The cDNA of the novel peptide, ranatuerin-2-AW (R2AW), was consistently cloned from the skin secretion of Amolops wuyiensis.The obtained nucleotide sequence is shown in Figure 1.The sequence was deposited in the GenBank with the accession number HF912236.The N-terminal 22 amino acid residues encoded a putative signal peptide, followed by an acidic spacer peptide domain of 17 amino acids.After a basic -KR-pro-peptide convertase cleavage site, a mature peptide domain of 29 amino acid residues was located at the C-terminus.Additionally, the sequences of R2AW, ranatuerin-2SRb [19], ranatuerin-2P-RA, and ranatuerin-2PLx [16] were aligned using the Uniprot database (available online: https://www.uniprot.org/(accessed on 9 March 2023)), as shown in Figure 2. The mature peptide domain of R2AW was a highly conserved ranatuerin-2-related peptide.Sequence alignment of R2AW and ranatuerin family peptide precursors derived from different frog species.The domains are divided into four parts, (1) the putative signal peptide, (2) the acidic spacer region, (3) the convertase processing site, and (4) the mature peptide.An asterisk (*) indicates that the residue in this position is fully conserved in the sequence alignment.A colon (:) indicates conservation between groups with strongly similar properties, while a period (.) indicates conservation between groups with weakly similar properties.

Peptide Design
Herein, we have proposed a progressive peptide design idea, and the physicochemical parameters of the peptides were evaluated by Heli-quest (Figure 3 and Table 1).To be specific, we first designed [Ser 23,29 ]R2AW to explore the effect of the intra-disulphide bond on the bioactivity of the ranatuerin-2 family.Serine residue possesses chemical properties and a structure similar to cysteine, with the only difference being the terminal groups of Additionally, the sequences of R2AW, ranatuerin-2SRb [19], ranatuerin-2P-RA, and ranatuerin-2PLx [16] were aligned using the Uniprot database (available online: https: //www.uniprot.org/(accessed on 9 March 2023)), as shown in Figure 2. The mature peptide domain of R2AW was a highly conserved ranatuerin-2-related peptide.Additionally, the sequences of R2AW, ranatuerin-2SRb [19], ranatuerin-2P-RA, and ranatuerin-2PLx [16] were aligned using the Uniprot database (available online: https://www.uniprot.org/(accessed on 9 March 2023)), as shown in Figure 2. The mature peptide domain of R2AW was a highly conserved ranatuerin-2-related peptide.indicates that the residue in this position is fully conserved in the sequence alignment.A colon (:) indicates conservation between groups with strongly similar properties, while a period (.) indicates conservation between groups with weakly similar properties.

Peptide Design
Herein, we have proposed a progressive peptide design idea, and the physicochemical parameters of the peptides were evaluated by Heli-quest (Figure 3 and Table 1).To be specific, we first designed [Ser 23,29 ]R2AW to explore the effect of the intra-disulphide bond on the bioactivity of the ranatuerin-2 family.Serine residue possesses chemical properties and a structure similar to cysteine, with the only difference being the terminal groups of indicates that the residue in this position is fully conserved in the sequence alignment.A colon (:) indicates conservation between groups with strongly similar properties, while a period (.) indicates conservation between groups with weakly similar properties.

Peptide Design
Herein, we have proposed a progressive peptide design idea, and the physicochemical parameters of the peptides were evaluated by Heli-quest (Figure 3 and Table 1).To be specific, we first designed [Ser 23,29 ]R2AW to explore the effect of the intra-disulphide bond on the bioactivity of the ranatuerin-2 family.Serine residue possesses chemical properties and a structure similar to cysteine, with the only difference being the terminal groups of hydroxyl and thiol on their respective sidechains.Therefore, a linear mutant was designed where the cysteines on positions 23 and 29 were substituted by serine so that it was unable to form a disulphide bridge.To further confirm whether the cyclic heptapeptide domain is essential for maintaining the function of R2AW, the truncated product R2AW(1-22) was synthesised by removing the Rana box at the C-terminus.Furthermore, considering that the amidation at the C-terminus would facilitate the antimicrobial activity and stability of AMPs, in addition to reducing the potential cytotoxicity [20][21][22], R2AW(1-22)-NH 2 was designed based on the sequence of truncated derivative R2AW .As shown in the helical wheel plots (Figure 3D), there are four lysine and two acidic aspartic acids, making the net charge +2 (HeliQuest calculates the net charge at pH = 7.4), and the positively charged residues were relatively dispersed.Considering that the cationicity and hydrophobicity play crucial roles in the function of AMPs [23], [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 was designed from R2AW(1-22)-NH 2 by substituting aspartic acids in positions 4 and 16 with lysine; meanwhile, a lysine on the hydrophobic side was replaced with leucine.To further explore the effect of hydrophobicity on the function of the peptide, [Trp 6,10 ]R2AW(1-22)-NH 2 was synthesised.The hydrophobic tryptophan residue with a bulky indole sidechain was reported to favour insertion into lipid bilayers [24,25].Therefore, two alanine residues on positions 6 and 10 were replaced with tryptophan residues.Table 1 summarises the physicochemical properties of R2AW and its five analogues, including peptide sequence, hydrophobicity, hydrophobic moment, and net charge.
Antibiotics 2024, 13, x FOR PEER REVIEW 4 of 23 hydroxyl and thiol on their respective sidechains.Therefore, a linear mutant was designed where the cysteines on positions 23 and 29 were substituted by serine so that it was unable to form a disulphide bridge.To further confirm whether the cyclic heptapeptide domain is essential for maintaining the function of R2AW, the truncated product R2AW(1-22) was synthesised by removing the Rana box at the C-terminus.Furthermore, considering that the amidation at the C-terminus would facilitate the antimicrobial activity and stability of AMPs, in addition to reducing the potential cytotoxicity [20][21][22], R2AW(1-22)-NH2 was designed based on the sequence of truncated derivative R2AW .As shown in the helical wheel plots (Figure 3D), there are four lysine and two acidic aspartic acids, making the net charge +2 (HeliQuest calculates the net charge at pH = 7.4), and the positively charged residues were relatively dispersed.Considering that the cationicity and hydrophobicity play crucial roles in the function of AMPs [23], [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH2 was designed from R2AW(1-22)-NH2 by substituting aspartic acids in positions 4 and 16 with lysine; meanwhile, a lysine on the hydrophobic side was replaced with leucine.To further explore the effect of hydrophobicity on the function of the peptide, [Trp 6,10 ]R2AW(1-22)-NH2 was synthesised.The hydrophobic tryptophan residue with a bulky indole sidechain was reported to favour insertion into lipid bilayers [24,25].Therefore, two alanine residues on positions 6 and 10 were replaced with tryptophan residues.Table 1 summarises the physicochemical properties of R2AW and its five analogues, including peptide sequence, hydrophobicity, hydrophobic moment, and net charge.The secondary structures of five analogues were predicted and the potential models are shown in Figure 4.A Ramachandran plot is commonly used to evaluate the quality and reliability of the predicted structure [26].The structure plausibility was assessed through ϕ (phi) and ψ (psi) angle distributions of the amino acid residues, classifying them into favoured (red), additionally allowed (yellow), generously allowed (pale yellow), and disallowed regions (white) [27,28].As displayed in Figure S1, there were few residues located in disallowed regions and more than 90% of the amino acids were plotted in the most favoured region, indicating the high quality of the predicted models.
As shown in Figure 5, the results of circular dichorism (CD) spectroscopy were in accordance with the Pepfold-3 secondary structure prediction.All tested peptides possessed an α-helical structure in 50% trifluoroethanol (TFE).

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Five Analogues of R2AW
The antimicrobial activities of five analogues of R2AW were investigated using six

Antiproliferative Activity of Designed Analogues of R2AW against Cancer Cells
The antiproliferative activity of designed analogues of R2AW was evaluated on five human cancer cell lines, including non-small-cell lung cancer (H838), human prostate carcinoma (PC-3), human neuronal glioblastoma (U251MG), human breast cancer cell (MCF-7), and colorectal carcinoma (HCT116), at concentrations ranging from 10 −9 M to 10 −4 M (Figure 9).Overall, the parent peptide R2AW inhibited the proliferation of cancer cell lines with half-maximal inhibitory concentration (IC 50 ) values ranging from 5.093 µM to 33.20 µM (Table 4), while [Ser 23,29 ]R2AW was the first designed peptide with the absence of disulphide bridges and this resulted in a slightly decreased activity against the growth of cancer cells.Also, it was found that the antiproliferative activity of truncated peptide R2AW   In the in vitro study, [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH2 had the best antibacterial activity among five designed peptides of R2AW.In this case, we applied an MRSA (NCTC 12493)-infected Galleria mellonella larvae model to further evaluate its in vivo antibacterial efficacy.As shown in Figure 8, the mortality of larvae that had been infected with MRSA declined substantially after the treatment with [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH2.Notably, no death of the larvae was observed in the presence of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH2.It was found that about 40% of larvae infected by MRSA survived at the dose of 10 mg/kg over five days.After a lower-concentration (5 mg/kg) treatment, the survival rate of infected larvae was around 10%.

Antiproliferative Activity of Designed Analogues of R2AW against Cancer Cells
The antiproliferative activity of designed analogues of R2AW was evaluated on five human cancer cell lines, including non-small-cell lung cancer (H838), human prostate carcinoma (PC-3), human neuronal glioblastoma (U251MG), human breast cancer cell (MCF-7), and colorectal carcinoma (HCT116), at concentrations ranging from 10 −9 M to 10 −4 M (Figure 9).Overall, the parent peptide R2AW inhibited the proliferation of cancer cell lines with half-maximal inhibitory concentration (IC50) values ranging from 5.093 µM to 33.20 µM (Table 4), while [Ser 23,29 ]R2AW was the first designed peptide with the absence of disulphide bridges and this resulted in a slightly decreased activity against the growth of cancer cells.Also, it was found that the antiproliferative activity of truncated peptide R2AW   The cell viability when using [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 on the human prostate cancer cell line, PC-3, was assessed by trypan blue exclusion assay.As shown in Figure 10, the killing effect of the peptide on cancer cells showed a dose-dependent pattern and reached a maximum within 6 h, indicating its strong efficiency.Specifically

Haemolytic Activities of R2AW and Its Designed Analogues
The haemolytic activity of five designed variants of R2AW was tested by the employment of a 2% (v/v) horse erythrocyte suspension.The results are shown in Figure 11.The parent peptide caused about 20% haemolysis up to 256 µM, while [Ser 23,29 ]R2AW and the truncated product R2AW(1-22)-NH2 showed lower haemolysis at around 10%.As for

Haemolytic Activities of R2AW and Its Designed Analogues
The haemolytic activity of five designed variants of R2AW was tested by the employment of a 2% (v/v) horse erythrocyte suspension.The results are shown in Figure 11.The parent peptide caused about 20% haemolysis up to 256 µM, while [Ser 23,29 ]R2AW and the truncated product R2AW(1-22)-NH 2 showed lower haemolysis at around 10%.As for R2AW , no haemolysis was observed after the removal of the Rana box without amidation.It is notable that the analogue [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 , with enhanced antibacterial and anticancer activities, caused no haemolysis at its maximum MBC value (16 µM), whereas [Trp 6,10 ]R2AW(1-22)-NH 2 , designed with the addition of two tryptophans, caused the strong haemolytic activity of approximately 20% at 2 µM.

Discussion
In this study, a novel peptide, R2AW, was isolated and characterised from the skin secretion of Amolops wuyiensis.Homology analysis using the BLAST programme and Uni-Prot database suggested that R2AW belongs to the ranatuerin-2 family.Similar to other peptides from this family (Figure 2), R2AW possessed a cyclic heptapeptide domain at the C-terminus, named a Rana box, whereas the primary structure was poorly conserved.The Rana box structure is found in most AMPs from Ranidae frogs.The function of this cyclic domain in helix-loop peptides remains ambiguous and arguable since it varies in different families.There are several statements about the role of the Rana box.On the one hand, positively charged residues concentrated in this domain provide a net charge.On the other hand, the Rana box could facilitate the helical structure and support protease resistance [11].In previous studies, researchers also pointed out that the Rana box was dispensable in the nigrocin family (e.g., nigrocin-HL) [18].However, the function of the cyclic heptapeptide fragment in the ranatuerin-2 family has not yet been thoroughly clarified.R2AW possessed low haemolytic activity and moderate antibacterial activity, similar to other peptides from the ranatuerin-2 family, such as ranatuerin-2PLx [16].Thus, five analogues were rationally designed to investigate the structural features of R2AW with the hope of improving both its antibacterial and anticancer activities.
The first analogue, [Ser 23,29 ]R2AW, was designed to have no intra-disulphide bond by substituting two cysteines at positions 23 and 29 with serine, which is relatively similar to cysteine.Currently, there is no agreement on whether the disulphide bond is indispensa-Figure 11.The haemolytic activities of R2AW and its analogues were evaluated using horse erythrocytes at peptide concentrations from 1 µM to 256 µM.Red blood cells treated with 0.1% Triton X-100 or PBS were regarded as positive control and negative control, respectively.The statistical significance was analysed using two-way ANOVA with Dunnett's multiple comparisons test in GraphPad Prism software by comparison with the negative control (* p < 0.05, ** p < 0.01, **** p < 0.0001).The error bar represents the SEM of nine replications.

Discussion
In this study, a novel peptide, R2AW, was isolated and characterised from the skin secretion of Amolops wuyiensis.Homology analysis using the BLAST programme and UniProt database suggested that R2AW belongs to the ranatuerin-2 family.Similar to other peptides from this family (Figure 2), R2AW possessed a cyclic heptapeptide domain at the C-terminus, named a Rana box, whereas the primary structure was poorly conserved.The Rana box structure is found in most AMPs from Ranidae frogs.The function of this cyclic domain in helix-loop peptides remains ambiguous and arguable since it varies in different families.There are several statements about the role of the Rana box.On the one hand, positively charged residues concentrated in this domain provide a net charge.On the other hand, the Rana box could facilitate the helical structure and support protease resistance [11].In previous studies, researchers also pointed out that the Rana box was dispensable in the nigrocin family (e.g., nigrocin-HL) [18].However, the function of the cyclic heptapeptide fragment in the ranatuerin-2 family has not yet been thoroughly clarified.R2AW possessed low haemolytic activity and moderate antibacterial activity, similar to other peptides from the ranatuerin-2 family, such as ranatuerin-2PLx [16].Thus, five analogues were rationally designed to investigate the structural features of R2AW with the hope of improving both its antibacterial and anticancer activities.
The first analogue, [Ser 23,29 ]R2AW, was designed to have no intra-disulphide bond by substituting two cysteines at positions 23 and 29 with serine, which is relatively similar to cysteine.Currently, there is no agreement on whether the disulphide bond is indispensable for bioactivity.In this study, it was found that [Ser 23,29 ]R2AW possessed a similar antibacterial activity in comparison to the parent peptide, while the anticancer activity was reduced slightly.This result suggested that the absence of disulphide bonds did not affect the antibacterial activity.In addition, it was observed that the substitution of cysteine with serine slightly decreased the helix content.Previous studies also showed that the reduction of intra-disulphide bonds, or replacing cysteine with serine into linear structures, would not affect the antibacterial activity but would reduce haemolysis [17,29], which is consistent with the results of this research.
Secondly, to confirm whether the cyclic heptapeptide domain is essential for maintaining the function of R2AW, the truncated product R2AW(1-22) was synthesised.Our study showed that the removal of the Rana box domain resulted in the reduction of helical content as well as bioactivities.This cyclic heptapeptide region adopts a helical loop-like fold stably constrained by the disulphide bridge, which is thought to stabilise the a-helical structure of AMPs.The loss of antimicrobial activity observed after removing the Rana box could be attributed to its crucial role in maintaining the helical stability and membrane binding affinity [11,30].
Furthermore, we also synthesised an amidated R2AW truncated derivative, R2AW(1-22)-NH 2 , for better comparison.The CD spectra revealed nearly identical helical content in R2AW  and R2AW(1-22)-NH 2 , suggesting minimal impact of C-terminal amidation on α-helical structure.As stated in the previous research [31,32], helical structure in AMPs is primarily sequence-and composition-dependent, with some AMPs maintaining helix content without C-terminal amidation due to intrinsic helix-forming ability.However, that does not negate the potential influence of amidated and unamidated C-terminuses on the functions of AMPs.Our results indicated that R2AW(1-22)-NH 2 retained antibacterial activity, which showed similarity to [Ser 23,29 ]R2AW.C-terminal amidation alters the surface charge and hydrophobicity of antimicrobial peptides, modulating their interactions with cell membranes [33].As reported, the amide group enhances adsorption and penetration abilities by forming hydrogen bonds and electrostatic interactions with membrane phospholipid molecules [20,22].Moreover, amidated peptides have a higher net charge compared to those with an unmodified C-terminus, potentially accounting for their increased activity [21].Therefore, amidation can compensate for activity loss due to Rana box removal, possibly by enhancing peptide-bacterial membrane interactions, thus surpassing the negative impact of the absence of this cyclic heptapeptide domain.In other words, the artificial elimination of the Rana box in the ranatuerin-2 family did not affect the antibacterial activity after C-terminal amidation.Notably, the antiproliferative activity of R2AW(1-22)-NH 2 decreased significantly with the absence of a Rana box.Although the reason is not apparent, it was speculated that this fragment affects the affinity of the peptide to cancer cells, making it indispensable.
It has been reported that the physicochemical properties of AMPs control their performance, like net charge and hydrophobicity, both of which have a significant impact on peptide potency [23].Therefore, a net-charge-and hydrophobicity-enhanced product, [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 , was designed based on the truncated sequence with the hope of optimising the antibacterial and anticancer activity.It was found that the ability of the modified product, [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 , to inhibit the growth of microorganisms was far more potent than the parent peptide, with MICs ranging from 2 µM to 8 µM towards six different bacteria.Therefore, it was further decided to explore the dynamic killing effect on bacteria.In general, the curves of time-killing and membrane permeability revealed that [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 killed bacteria rapidly through membrane permeabilization.It also acted in a concentration-dependent manner.As shown in Figures 4D and 6D, there was a rising tendency of killing in E. coli and P. aeruginosa in the presence of 0.5 × MIC and 1 × MIC of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 , respectively.This phenomenon can be explained by the different values of MIC and MBC.The concentration of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 to kill and inhibit the growth of E. coli was the same, but that was not the case for P. aeruginosa.Moreover, the concentration of surviving bacteria in the sample treated with the peptide was far too low to be observed in the viable cell counts.According to several publications, a higher density of peptide will accumulate on the lipid membrane, acting as a carpet, thus causing membrane lysis.However, a smaller number of smaller-sized toroidal pores were most likely created at lower peptide concentrations [34].In this case, [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 was suspected of acting in the same way.In other words, AMPs, in the absence of the target-specific receptor on the surface of microorganisms, displayed a lower possibility of developing resistance compared to conventional antibiotics.Regarding the promising antibacterial activity of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 , the in vivo efficacy was accessed in the MRSA-infected Galleria mellonella larvae (waxworm) model [35].[Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 could decrease the mortality rate of infected waxworms, but it was less effective than existing antibiotics, which could be attributed to its low stability.Furthermore, it was indicated that [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 has no toxicityin vivo, which provided a guideline for further in vivo research.Apart from the excellent antibacterial activity, the potency of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 against the proliferation of cancer cells was evaluated.Generally, [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 showed significance against five human cancer cell lines, especially human prostate cancer cells, at a concentration of 10 µM.According to the trypan blue exclusion assay, [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 only induced partial cytotoxicity at 25 µM.It is reasonable to speculate that there is another mechanism whereby the antiproliferation effects on cancer cells can be achieved without cell membrane disruption, which needs to be further evaluated.Also, the cytotoxicity of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 should be assessed on normal human cells in future work.In summary, the results showed that the function of R2AW was enhanced significantly after increasing the net charge and hydrophobicity by swapping two acidic amino acids, aspartic acid, with lysine and introducing a leucine residue.To some extent, peptides with positive charges can electrostatically bind with the negatively charged membranes of microorganisms [36,37].Specifically, the anionic cell wall teichoic acids (WTAs) and lipoteichoic acids (LTAs), attached to the peptidoglycans in Gram-positive bacteria and Gram-negative bacteria, have an outer membrane composed of lipopolysaccharide, making them preferable for AMP binding [38].In the same way, the membrane of a cancer cell typically has a negative charge because of the high number of anionic molecules like phosphatidylserine, O-glycosidic mucins, and sialylated gangliosides, which could facilitate this interaction [39].Regarding hydrophobicity, generally, the property of hydrophobicity in AMPs determines the possible extent of penetration of the lipid bilayer leading to membrane disintegration [40].
To further explore the effect of hydrophobicity on the function of the peptide, [Trp 6,10 ]R2AW(1-22)-NH 2 was synthesised by substituting two alanine residues with tryptophan residues.The results indicated that the antibacterial activity of [Trp 6,10 ]R2AW(1-22)-NH 2 was slightly decreased compared to [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 .Though [Trp 6,10 ]R2AW(1-22)-NH 2 exhibited the most potent negative effects on the growth of cancer cells, its strong haemolysis limited applications in the clinic.Experiments on the membrane simulation model demonstrated that higher hydrophobicity produces both efficiency and cytotoxicity [41].Nevertheless, a previous study also indicated a threshold hydrophobicity at which better bioactivity could be achieved [42].The results here showed that the substitution with tryptophan led to excessive hydrophobicity, which produced an elevated potency against pathogens but also caused damage to host cells.
In conclusion, this study indicated that the antibacterial activity of ranatuerin-2 family peptides could be enhanced via the substitution of acidic amino acids with positively charged lysine residues and the introduction of the leucine on the hydrophobic surface after the removal of the Rana box.Meanwhile, the designed peptide [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 , with significantly improved activity and a potential in vivo therapeutic effect against MRSA, could be a rational subject for further study toward clinical applications.

Acquisition of Frog Skin Secretion
Specimens of the frogs, Amolops wuyiensis (n = 3), were obtained from a commercial supplier in the United Kingdom.All the frogs were adults, and they were kept for at least four weeks in a specialised tropical amphibian facility at Queen's University Belfast, where they were fed multivitamin-loaded crickets every 2 days and maintained on a 12 h/12 h day and night cycle at 25 • C. The frog skin secretions were collected through gentle electrical stimulation (5V, 100 Hz, and 140 ms pulse width).Subsequently, the stimulated viscous secretions were washed into a cold beaker with deionized water, snap frozen in liquid nitrogen, and then lyophilized.Before analysis, the samples were stored at −20 • C.

'Shotgun' Cloning and Sequencing of Ranatuerin-2-AW Precursor-Encoding cDNA
Five mg of lyophilised Amolops wuyiensis skin secretion was dissolved in 1 mL of Lysis/Binding buffer.A Dynabeads ® mRNA DIRECT™ Kit (Dynal Biotech, Merseyside, UK) was used for mRNA isolation.The cDNA library construction was performed using Clontech SMARTer ® RACE 5 /3 Kit (Takara Bio, USA, Inc., San Jose, CA, USA).A nested universal primer (NUP) and a degenerate sense primer (S1: 5 -GAWYYAYYHRAGCCYAAADATG-3 ; W = A + T, Y = C + T, H = A + C + T, R = A + G, D = A + G + T) were used.The cDNA ends were rapidly amplified by a PCR thermal cycling system with repeated denaturation, annealing, and extension.The products were analysed by gel electrophoresis and then purified using a Hi-Bind DNA mini-column (Omega Bio-Tek, Norcross, GA, USA).The process of DNA ligation was performed using a pGEM-T Easy Vector System (Promega Corporation, Madison, WI, USA).After that, the recombinant plasmid DNA was cloned in JM109 high-efficiency competent cells (Promega, Madison, WI, USA).A Big Dye ® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was used for sequencing reaction and the products were analysed using an ABI3730 automated sequencer (Applied Biosystems, Foster City, CA, USA).

Purification and Characterisation of R2AW and Its Designed Analogues
The crude peptides were purified using a reverse phase HPLC system (Waters ® , Milford, MA, USA) that consisted of Waters 1500 Series pumps, a Waters 2489 UV/Visible Detector, and a Phenomenex Aeris 5 µm Peptide Xb-C18 Column 250 × 21.2 mm.The molecular masses of the purified products were obtained and confirmed by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (Perspective Biosystems, Framingham, MA, USA) (Supplemented in Figures S2 and S3).A total of 10 mg/mL of the α-cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix solution.

Circular Dichroism Spectra
The secondary structures of R2AW and its five analogues were investigated using a JASCO-815 CD spectrometer (JASCO Inc., Tokyo, Japan).Each peptide was dissolved in 50% TFE (v/v in 10 mM NH 4 Ac (pH = 7)) to a final concentration of 50 µM and then 200 µL of each peptide solution was loaded into a 1 mm path length curette.Samples were analysed within the range of 190 to 250 nm at room temperature at a scanning speed of 100 nm/min, a bandwidth of 1 nm, and a data pitch of 1 nm.

Determination of MICs and MBCs
The MIC and MBC assays were used to assess the inhibitory activities of all 5 analogues of R2AW on the growth of planktonic microorganisms.The MIC is the lowest concentration that prevents the viable growth of bacteria.The MBC is the lowest concentration that results in bacterial death.Six microorganisms were used to detect the antimicrobial activity of peptides, including Gram-positive bacteria S. aureus (NCTC 10788), MRSA (NCTC 12493), Enterococcus faecalis (E.faecalis) (NCTC 12697), Gram-negative bacteria E. coli (ATCC 8379), Klebsiella pneumoniae (K.pneumoniae) (ATCC 43816), and Pseudomonas aeruginosa (P.aeruginosa) (ATCC 9027).According to the assay described previously [44], the microorganisms were inoculated at 37 • C overnight and diluted to 5 × 10 5 CFU/mL after reaching the logarithmic growth period.The peptide stock solution at the concentration of 512 × 10 2 µM was prepared and diluted to 100 µM by a 2-fold dilution.One µL of each concentration of peptide solution was incubated with 99 µL of bacterial subculture in the 96-well plate for 18 h at 37 • C. The absorbance was determined at a wavelength of 550 nm using a Synergy HT plate reader (Bio Tek, Washington, DC, USA).The peptide concentration that resulted in invisible bacteria in the 96-well plate was determined as the MIC.As for those wells that were clearly visible, 20 µL of the solution was dropped onto the Mueller Hinton agar (MHA) dish and then incubated at 37 • C overnight.The lowest concentration with no colony growth was determined as the MBC.

Antibiofilm Assays
As a previous study [45] described in detail, the ability of R2AW and its analogues to inhibit and eradicate the formation of biofilm was investigated.The MBIC is the lowest concentration which inhibits the formation of biofilm.The MBEC is the minimum concentration that eradicates biofilm.Six bacterial strains were used, as described above.Briefly, the bacteria were cultured and then diluted to 5 × 10 5 CFU/mL.In MBIC assays, 1 µL of each concentration of R2AW and its analogues (ranging from 512 × 10 2 µM to 100 µM) was mixed with 99 µL of diluted bacterial suspension in a 96-well plate for 16-20 h at 37 • C. As for MBEC assays, 100 µL of cultured bacteria were seeded in a 96-well plate and incubated on the shaking incubator at 37 • C overnight for biofilm formation.Subsequently, each well was rinsed twice with 100 µL sterile PBS to remove planktonic cells, and then replaced with 100 µL of each concentration (ranging from 512 µM to 1 µM) of peptides.After 24 h incubation, the biofilm was washed twice with 100 µL of PBS and fixed with methanol.Next, the same volume of 0.1% crystal violet solution (Sigma-Aldrich, Gillingham, UK) was added to each well to stain the biofilm.After that, 100 µL of 30% of acetic acid (Sigma-Aldrich, Gillingham, UK) was transferred to each well for dissolution.The solutions were transferred into a new 96-well plate and the absorbances of wells was monitored using a Synergy HT plate reader set to 595 nm.

Bacterial Cell Membrane Permeability Assays
The effect of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 on membrane integrity of S. aureus (NCTC 10788), E. coli (ATCC 8739), MRSA (NCTC 12493), and P. aeruginosa (ATCC 9027) was studied by applying SYTOX Green nucleic acid stain (Thermo Fisher Scientific, Waltham, MA, USA).These four bacteria were inoculated into a TSB medium and grown to the logarithmic phase.The supernatant was decanted after centrifugation (1000× g, 10 min, 4 • C).The bacteria were washed with 5% TSB in 0.85% NaCl solution and resuspended until a 1 × 10 8 CFU/mL density was reached and assessed by measuring the OD value (0.7) at wavelength 590 nm.Forty µL of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 at concentrations of 4 × MIC, 2 × MIC, and 1 × MIC was added to a black 96-well plate.Meanwhile, 10 µL of diluted SYTOX green-fluorescent nucleic acid stain (50 µM, Life Technologies, Renfrew, UK) was transferred into each well.Next, 50 µL of the bacterial suspension (1 × 10 8 CFU/mL) was transferred into each well.The plate was analysed by the Synergy HT plate reader using a 2 h kinetic programme with excitation at 485 nm and emission at 580 nm.According to a previous publication, the in vivo antibacterial activity of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 was assessed using the larva of Galleria mellonella with minor revision [46].The waxworms (Livefood UK Ltd., Rooks Bridge, UK) were weighed, then larvae (250 ± 50 mg) were picked and used at 10 per plate.The waxworms were injected with 10 µL of MRSA (NCTC12493) suspension (1 × 10 7 CFU/mL) that was prepared in sterilised PBS.After 1 h of infection, each waxworm was administered 10 µL of either 5 mg/kg or 10 mg/kg peptide solution.The same injection volume of PBS and 50 mg/kg vancomycin were regarded as negative and positive controls, respectively.Each group contained 10 larvae and the numbers of survivors were recorded every 12 h for five days.

Anticancer MTT Assays
Five human cancer cell lines of non-small-cell lung cancer H838, human prostate carcinoma PC-3, human neuronal glioblastoma U251MG, human breast cancer cell MCF-7, and colorectal carcinoma HCT116 were used to detect the antiproliferative abilities of R2AW and its five analogues.According to the procedures in a previous article [47], cells (8000 cells/well) were seeded into the 96-well tissue culture plates and incubated at 37 • C with 5% CO 2 overnight.The solutions of R2AW and its analogues at concentrations of 10 −4 M to 10 −9 M were prepared in the serum-free medium.Next, cells in each well were treated with 100 µL of each peptide solution for 24 h.Subsequently, 10 µL of 5 mg/mL MTT (3-(4, 5-Dimethylthiazol-2-yl-)-2, 5-Diphehyltetrazolium Bromide) was added to each well followed by 37 • C incubation for 2 h.After that, the liquid in each well was removed and 100 µL of DMSO was added to dissolve the purple formazan.The plate was placed on a shaking incubator for 10 min, and the absorbance was detected at 550 nm using a Synergy HT plate reader (Biotech, Minneapolis, MN, USA).

Trypan Blue Exclusion Assays
In the MTT assay, [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 showed the best antiproliferative activity against human prostate cancer cells, PC-3.In this context, the cytotoxicity of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 on PC-3 cells was determined by trypan blue exclusion assay [43].Cells (2 × 10 5 cells/well) were seeded into a 12-well plate and incubated at 37 • C with 5% CO 2 overnight.After 4 h of starvation, 500 µL of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 at the concentrations of 50 µM, 25 µM, 10 µM, and 5 µM was added to each well and incubated at 37 • C for 2 h, 6 h, and 24 h.At each time point, the cell suspension of each well was made and transferred to the 1.5 mL tube for centrifugation (380× g, 5 min, 18 • C).After removing the supernatant, 400 µL of PBS was added to resuspend the cell pellet.Next, 10 µL of cell suspension was mixed with the same volume of trypan blue (0.4%, Gibco, Brooklyn, NY, USA).Then, 10 µL of the mixture was loaded into a haemocytometer for cell counting.The percentages of viable cells and dead cells were calculated by counting the unstained and stained cells, separately.

Haemolysis Assays
The haemolytic activity of R2AW and its five designed analogues on mammalian erythrocytes was evaluated using 2% defibrinated horse blood (TCS Biosciences Ltd., Buckingham, UK) as per a previous report [48].Two mL of fresh horse blood was transferred into a 50 mL tube and then washed with sterilised PBS.The supernatant was removed after centrifuging (930× g, 5 min).The centrifugation procedure was repeated until the supernatant was clear.After washing, the red blood cell pellet was diluted with PBS to achieve a 4% (v/v) erythrocyte suspension.The concentrations of R2AW and its designed derivatives were prepared from 512 µM to 2 µM by two-fold dilution in sterilised PBS.One hundred µL of red blood cell suspension was added into a 1.5 mL tube treated with the same volume of each concentration of peptide and incubated at 37 • C for 2 h.The red blood cell suspensions treated with 0.2% Triton X-100 and with PBS were regarded as positive control and negative control.After centrifugation at 930× g for 10 min, 100 µL of suspension was transferred to a 96-well plate, and the absorbance was obtained by use of a Synergy HT plate reader.The degree of haemolysis could be calculated using the following formula: Haemolysis = (absorbance (experimental groups) − absorbance (negative control))/(absorbance (positive control) − absorbance (negative control)) × 100%.

Figure 2 .
Figure 2. Sequence alignment of R2AW and ranatuerin family peptide precursors derived from different frog species.The domains are divided into four parts, (1) the putative signal peptide, (2) the acidic spacer region, (3) the convertase processing site, and (4) the mature peptide.An asterisk (*)indicates that the residue in this position is fully conserved in the sequence alignment.A colon (:) indicates conservation between groups with strongly similar properties, while a period (.) indicates conservation between groups with weakly similar properties.

Figure 2 .
Figure 2. Sequence alignment of R2AW and ranatuerin family peptide precursors derived from different frog species.The domains are divided into four parts, (1) the putative signal peptide, (2) the acidic spacer region, (3) the convertase processing site, and (4) the mature peptide.An asterisk (*)indicates that the residue in this position is fully conserved in the sequence alignment.A colon (:) indicates conservation between groups with strongly similar properties, while a period (.) indicates conservation between groups with weakly similar properties.

Figure 2 .
Figure 2. Sequence alignment of R2AW and ranatuerin family peptide precursors derived from different frog species.The domains are divided into four parts, (1) the putative signal peptide, (2) the acidic spacer region, (3) the convertase processing site, and (4) the mature peptide.An asterisk (*)indicates that the residue in this position is fully conserved in the sequence alignment.A colon (:) indicates conservation between groups with strongly similar properties, while a period (.) indicates conservation between groups with weakly similar properties.

Figure 6 .
Figure 6.The kinetic time-killing curves of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH2 against (A) S. aureus, (B) MRSA, (C) E. coli, and (D) P. aeruginosa at concentrations of 0.5 × MIC, 1 × MIC, 2 × MIC.The peptide was added at 0 min and monitored until 180 min.Vancomycin was used as a traditional antibiotic control for Gram-positive bacteria, and colistin was used as a peptide control for Gram-negative bacteria.The CFU/mL of E. coli in the presence of 0.5 × MIC of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH2 at 180 min was compared with that at 90 min by t-test.The CFU/mL of P. aeruginosa in the presence of 1 × MIC of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH2 at 180 min was compared with that at 90 min using a t-test.The level of significance was ** p< 0.01.

Figure 6 .
Figure 6.The kinetic time-killing curves of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 against (A) S. aureus, (B) MRSA, (C) E. coli, and (D) P. aeruginosa at concentrations of 0.5 × MIC, 1 × MIC, 2 × MIC.The peptide was added at 0 min and monitored until 180 min.Vancomycin was used as a traditional antibiotic control for Gram-positive bacteria, and colistin was used as a peptide control for Gramnegative bacteria.The CFU/mL of E. coli in the presence of 0.5 × MIC of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 at 180 min was compared with that at 90 min by t-test.The CFU/mL of P. aeruginosa in the presence of 1 × MIC of [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 at 180 min was compared with that at 90 min using a t-test.The level of significance was ** p< 0.01.

23 Figure 10 .
Figure 10.The cell viability when using [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH2 was evaluated by trypan blue exclusion assay.Human prostate cancer cells PC-3 were treated with the peptide at concentrations of 50 µM, 25 µM, 10 µM, and 5 µM for 2, 6, and 24 h, respectively.The error bars represent the SEM of six replications.The percentage of cell viability was obtained by calculating the ratio of survival cells to total cells.

Figure 10 .
Figure 10.The cell viability when using [Lys 4,19 , Leu 20 ]R2AW(1-22)-NH 2 was evaluated by trypan blue exclusion assay.Human prostate cancer cells PC-3 were treated with the peptide at concentrations of 50 µM, 25 µM, 10 µM, and 5 µM for 2, 6, and 24 h, respectively.The error bars represent the SEM of six replications.The percentage of cell viability was obtained by calculating the ratio of survival cells to total cells.

Antibiotics 2024 , 23 Figure 11 .
Figure11.The haemolytic activities of R2AW and its analogues were evaluated using horse erythrocytes at peptide concentrations from 1 µM to 256 µM.Red blood cells treated with 0.1% Triton X-100 or PBS were regarded as positive control and negative control, respectively.The statistical significance was analysed using two-way ANOVA with Dunnett's multiple comparisons test in GraphPad Prism software by comparison with the negative control (* p < 0.05, ** p < 0.01, **** p < 0.0001).The error bar represents the SEM of nine replications.

Table 1 .
Structural parameters of R2AW and its analogues.Heli-quest calculates the net charge at pH = 7.4.

Table 2 .
MICs and MBCs of R2AW and its analogues against different microorganisms.